J. M. Vincent

The influence of the host on competition amongst clover root-nodule bacteria.

SUMMARY: Serological typing has been used to study competition between five strains of Rhizobium trifolii in growth and nodulation with four species of host. Although a proportion of non-reacting antigenic variants appears to have been encountered, the serological method remains uniquely suitable for studies of this kind. Irrespective of host species or level of inoculum the population of bacteria in the tube of seedling agar approached a maximum of approximately 4 x 106 viable cells per ml. No consistent relationship was found between host species and the proportion of the several strains of rhizobia in the population outside the plant. The relative frequency of strains was, however, markedly affected by level of total inoculum and time of growth after inoculation. Some strains grew better at first but reached a lower maximum than others which fared better at higher population levels, whether from a heavier inoculum or prolonged growth. The population on the root surface itself failed to reflect any marked host influence in the balance between strains. The proportions of strains found in the nodules were unrelated to their representation in the roots external environment. Each host exercised a specific selective effect in this regard.

Influence of Calcium and Magnesium on the Growth of Rhizobium

SUMMARY: Growth of Rhizobium trifolii in a defined medium reflected the supply of Ca2+ and Mg2+ (subsequently Ca and Mg, respectively) in distinctive fashion. Deficiency of Ca, in the presence of sufficient Mg, caused reduction in growth rate, the level of maximum growth and the proportion of viable cells. Such Ca-deprived cells were markedly swollen and vacuolated. On the other hand, although shortage of Mg (Ca sufficient) was without effect on growth rate down to the lowest concentration at which growth occurred, maximum growth and the proportion of viable organisms were markedly decreased. Mg-deficient organisms were appreciably elongated. Signs of Ca deficiency became apparent at less than 0.025 mM, and Mg deficiency at less than 0.1 mM, most markedly in the range below 0.5 mM. Additionally there was a need for total divalent cations of the order of 0.4-0.6 mM. This could be met by either Ca or Mg provided both were sufficient for their maximum specific effect.

Taxonomically significant group antigens in Rhizobium.

SUMMARY: Earlier studies of the antigenic determinants of Rhizobium were almost entirely concerned with agglutinogens and revealed considerable strain specificity, the recognition of which was enhanced by a distinction between flagellar and somatic antigens (Bushnell & Sarles, 1939; Vincent, 1941, 1942; Kleczkowski & Thornton, 1944; Purchase, Vincent & Ward, 1951; Means, Johnson & Date, 1964). In some cases this specificity depended on complex patterns of shared antigens, in others on complete non-cross reactivity between strains that belonged to the same species. Agglutination could not therefore be depended on for the recognition of species or groups of species of rhizobia. On the other hand, the technique has proved valuable for the definition of serotypes and the labelling of strains used for experimental purposes.

Calcium in cell walls of Rhizobium trifolii.

SUMMARY: The cell walls from Rhizobium trifolii, grown under both ‘normal’ and ‘calcium-deprived’ conditions, were analysed in an attempt to detect a chemical cause for the apparent weakness or looseness of the walls in calcium-deprived organisms. The organic components typical of Gram-negative cell walls were present in normal and calcium-deprived cells. The latter concentrated most of the small amount of available calcium in the walls, which, however, contained only 60% of that present in the walls of normal organisms. Magnesium was not able to substitute for calcium as a wall component.

Extracellular Polysaccharides of Rhizobium

SUMMARY: The extracellular polysaccharides of seventeen strains (four species) of Rhizobium were studied chromatographically. The seven strains from lucerne (R. meliloti) produced polysaccharides which contained glucose but consistently lacked glucuronic acid. The remaining ten strains, which were isolated from pea (R. leguminosarum), clover (R. trifolii) and bean (R. phaseoli), produced a polysac-charide in which both glucose and glucuronic acid were demonstrated. These latter strains could be grouped into three types according to their glucose: glucuronic acid ratio, but such groups did not correspond with the species.

Some features of the fine structure and chemical composition of Rhizobium trifolii.

SUMMARY: The conspicuous, large, high refractive index, sudanophilic granules of Rhizobium trifolii appeared to be aggregations of polymeric β-hydroxybutyric acid, probably closely associated with the cytoplasm. They became more conspicuous as the organism aged, provided that carbohydrate was in excess. A well-grown culture contained 40–50% polymer, based on cell dry weight. Relatively large cytoplasmic granules (50–80 mμ) were a feature of this organism whether in fixed and sectioned cells or in material shadowed after mechanical disintegration. Classical ‘double’ (? lipoprotein) membranes were demonstrated both for the cell wall and the cytoplasmic membrane. Carefully fixed and embedded material often showed an accumulation of material between the two double membranes, especially at one or both ends, without any evidence of gross damage in the sectioned organisms.

The Effect of Calcium Nutrition on the Production of Diffusible Antigens by Rhizobium trifolii

SUMMARY: Rhizobium trifolii grown in a defined medium deficient in calcium yielded additional diffusible antigens as compared with organisms receiving calcium or strontium. The two kinds of organisms were indistinguishable in respect of their somatic agglutinogens. The same additional precipitinogens were released from Ca-adequate bacteria by mechanical disintegration, freezing, drying, lysozyme or chloroform. Their occurrence in the untreated Ca-deficient bacteria is attributed to autolysis of these more fragile bacteria. The precipitinogen (‘a’ band) that was common to the Ca-adequate and Ca-deficient bacteria was not shared by a second strain of R. trifolii nor by 2 strains of R. meliloti. This ‘a’ antigen was heat stable and appeared to be a component of the cell wall, from which it could be separated in diffusible form by formamide extraction of whole bacteria, trypsin digestion of broken bacteria and by mechanical disintegration of prepared cell walls. Neither of the additional antigens (‘b’, ‘c’) was related to agglutination; both were found in Ca-deficient or mechanically broken bacteria of the other strain of R. trifolii; ‘c’ only was similarly shared with R. meliloti. The ‘b’ band antigen appeared to be an intracellular component and was heat labile. Like ‘a’, ‘c’ was heat stable and associated with cell wall, but evidently was not ordinarily exposed, although readily released by lysis or mechanical breakage. The extracellular polysaccharide was neither antigenic nor haptenic, but the preparation obtained from Ca-deficient and mechanically broken Ca-adequate organisms was persistently associated with a co-precipitating antigenic component (chiefly ‘a’ band).

The Somatic Antigens of Two Strains of Rhizobium trifolii

SUMMARY: Strain-specific lipopolysaccharide extracted by hot phenol from two strains of Rhizobium trifolii was fully antigenic in rabbits with an injection schedule using Freunds adjuvant, and highly active in gel diffusion. A minor second diffusion line was considered to be due to a smaller fragment of the main molecule, since the antigen was converted almost entirely to this form by sodium dodecylsulphate and such disaggregated material absorbed antibodies to both forms of the antigen. Chemically the lipopolysaccharide showed some features related to those of the Enterobacteriaceae; for example, the presence of firmly bound lipid, 2-keto-3-deoxyoctonate, glucose, man-nose, fucose and, in one strain, a heptose. However, the lipopolysaccharide of R. trifolii was unusual in its very low phosphorus content and the presence of glucuronic acid. One strain diverged even further in its high content of carbohydrate, its lack of a heptose, its anionic behaviour and the gelatinous nature of some preparations; these properties might relate to the presence of a capsule-like structure which has been observed in old cultures of this strain.